Field of the Invention
The present invention relates to a pathogen-inducible synthetic promoter which is suitable for regulating the transcription of a nucleic acid and includes a minimal promoter. Further, the present invention relates to a transgenic plant cell as well as transgenic plants. The present invention further concerns a process for producing a pathogen resistant plant.
Description of the Related Art
Various processes are known for creating plants which are resistant against pathogens such as fungi, virus, bacteria and nematodes. One of these processes employs the hypersensitive reaction (HR) of the plant, wherein the development of necrosis occurs at the location of direct contact between pathogen and plant. As a consequence of the HR a broad spectrum of pathogen defense mechanisms are triggered in adjacent cells, which prevent the further propagation of the pathogen in the plant tissue.
The HR can occur after expression of effector genes, such as for example avirulence genes of the pathogen and interaction with the product of a corresponding resistance gene (R-gene). The R-gene can herein already be present in the plant or, as the case may be, may be introduced by gene technology methods into the respective plant genome (Stuiver et al. 1998, Keller et al., 1999, Belbahri et al., 2001). Besides this, an over-expression or autoactivation of R-genes can lead to triggering of a HR (Tao et al., 2000, Tang et al., 1999, Bendahmane et al., 2002, Howles et al., 2005). By the over-expression of a R-gene a threshold is exceeded, which leads to initiation of a signal cascade, which conventionally is only initiated upon the presence of the pathogen or as the case may be the avirulence gene product. By triggering or activating this cascade a broad effective pathogen resistance can be achieved (Oldroyd and Staskawicz, 1998, Tang et al., 1999, Tao et al., 2000, Howles et al., 2005). Those R-genes are characterized as autoactive R-genes which are modified to the extent that for initiation of the signal cascade the presence of the pathogen/avirulence gene product is not necessary and at the same time, a reduced level of expression in comparison to the non-modified form is sufficient in order to achieve initiation of the signal cascade.
Stuiver et al. (1998) were able to show that the transformation of the avr9-gene from the phytopathogenic fungi Cladosporium fulvum under the control of the pathogen inducible Gst1-promoter from the potato in tomato plants, which carry the corresponding Cf9-gene, brought about a broad effective fungi resistance. A resistance against the oomycete Phytophthora parasitica var nicotianae could be achieved in Nicotiana tabacum after either the elicitor cryptogen from P. cryptogea or the bacterial elicitor popA from the phytopathogenic bacterium Ralstonia solanacearum was transformed in N. tabacum. Both genes were under the control of the pathogen inducible promoter hsr203J from N. tabacum (Keller et al., 1999, Belbahri et al., 2001).
The system of the HR triggering requires a stringent control of the expression of the effector gene at the location of the infection. In the case of uncontrolled expression, the expression of the effector gene causes negative effects on plant growth and therewith on the harvesting of horticultural plants (Stuiver and Custers, 2001). A controlled expression can however occur by the selection of suitable pathogen inducible promoters. These should, however, no expression or only a small expression under conditions of non-infestation, however, in the case of infection, cause a significantly higher expression at the location of the infection. After transformation from two different autoactive forms of the L6 rust resistance gene from flax (Linum usitatissimum) in flax under the control of the natural Fis1 promoters inducible by rust from flax, two phenotypes could be observed. On the one hand, normal growth plants, which showed no improved resistance against pathogens, and on the other hand, dwarf plants, with a broad pathogen resistance (Howles et al., 2005). These results show that, depending upon the employed form of the autoactive R-gene, the result could be a promoter activity which already lies above the threshold for induction of the signal cascade, while in the phenotypically unremarkable plants the induction of the Fis1-promoters is not sufficient in order to achieve this threshold. The specificity of the natural Fis1-promoters thus is not sufficient in order to achieve the broad effective pathogen resistance without negative effects on the plant growth.
Natural pathogen inducible promoters frequently show a non-specific activity and are activated by numerous stimuli, so that their use for the expression of the above-described effector genes is not practical, since a HR-triggering could also occur under non-infection conditions. This “leakiness” of the promoters leads to an impairment of plant growth and thus to a reduction of the harvest yield of horticultural crops. For this reason synthetic promoters were developed, which contain the sequence motives (cis-regulatory elements) from natural, pathogen inducible promoters, which are relevant for pathogen induction. Sequence motives for other stimuli are, in contrast, removed. The cis-regulatory elements are cloned upstream of the minimal promoter, whereby a functional promoter is produced, which exhibits an elevated specificity in comparison to the natural promoters, which were isolated from the respective cis-regulatory elements (Rushton et al., 2002). As minimal promoter for dicotyledonous plants the region −46 through +8 of the 35S-gene of the Cauliflower Mosaic Virus was employed. Besides this, the use of a minimal promoter from a natural promoter, out of which the respective cis-regulatory element was cloned, are known (Perl-Treves et al., 2004). For monocotyledonous plants, the use of the minimal promoter from the Actl-gene of rice is described (Lü et al., 2000).
Although the described synthetic promoters are an improvement over the natural promoters, these however show background activity even under non-infection conditions. These background activities vary among individual plant types. Thus, in all plant types examined until now a pathogen inducibility could be determined, however the strength of the induction and the absolute activity of the promoters vary. In the case of a too-strong background activity in non-infected tissue, then, only a small pathogen inducibility could be determined as quotient of the promoter activity in the infected tissue divided by the promoter activity in the non-infected tissue.
Until now, only the employed cis-regulatory elements were considered responsible for the level of the background activity of a synthetic promoter. These have a large influence on the strength of the promoter (Rushton et al., 2002). Little investigated until now was the influence of the minimal promoter. According to the literature the minimal promoter has only a very small influence on the regulation of the promoter activity (Singh, 1998). Bhuliar et al. (2003) could however detect a clear reduction of the promoter activity of the 35S-promoter when the minimal promoter (−46 through +1) was exchanged with heterologous plant minimal promoters. These differences lead back to the different sequences of the TATA-boxes, while, according to their opinion, the flanking regions of the TATA-box of the minimal promoter are not relevant for the promoter activity.
It is thus the task of the present invention to provide a pathogen inducible synthetic promoter with a small background activity.